C. elegans is a powerful and well-established model organism used to investigate basic and conserved molecular mechanisms underlying development, apoptosis, cellular proliferation, aging, and neurological disease. However, targeted mutagenesis of endogenous chromosomal genes or site-specific insertion of transgenes by gene targeting methods used in other organisms is highly inefficient in C. elegans and, therefore, inaccessible to most researchers. However, recent work in Drosophila, plants, and mammalian cells has shown that introduction of a double-stranded break (DSB) at a sequence of interest can stimulate rates of gene targeting by many orders of magnitude. In these studies, DSBs were introduced at specific genomic sequences using zinc finger nucleases (ZFNs), artificial restriction endonucleases that can be engineered to cleave specific target DNA sequences. This exploratory R21 proposes to develop ZFN-stimulated gene targeting for use in C. elegans. The long-terms goals of this project include establishing optimal techniques for gene targeting and gene inactivation in C. elegans and establishing strategies to provide designer Zn-finger proteins for gene targeting for the broader academic research community. The specific aims of this proposal are: (1) to test whether ZFNs can be used to introduce a DSB into an integrated GFP reporter gene; and (2) to test whether ZFN-enhanced gene targeting can be used to create specific mutations or to introduce transgenes (e.g. GFP) into specific endogenous genes in the C. elegans germline. The proposed studies will provide a powerful and novel genetic technique for C. elegans-based research. Accelerating and expanding C. elegans research will directly increase the speed and power of the analyses that can be carried in this well-established model organism. Development of efficient gene targeting techniques will permit C. elegans researchers to establish more accurate models of human disease by inserting specific changes into C. elegans homologs of human disease-related proteins. We expect that the development of ZFN-based gene targeting techniques will result in a more rapid understanding of basic mechanisms underlying human disease. Public Health Relevance Modeling human diseases in other vertebrate or invertebrate systems requires technologies to precisely alter gene structure. Investigations of genetic and developmental processes in C. elegans have provided important insights, however, the lack of a tractable method for manipulating endogenous gene structure has been a limitation. Development of the Zinc-Finger Nuclease technology for manipulating endogenous sequences in C. elegans will significantly augment this already powerful system by allowing more precise modeling of human genetic diseases. [unreadable] [unreadable] [unreadable]